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- PDB-9fjf: Lysosomal transporting complex of beta-glucocerebrosidase (GCase)... -

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Entry
Database: PDB / ID: 9fjf
TitleLysosomal transporting complex of beta-glucocerebrosidase (GCase) and lysosomal integral membrane protein 2 (LIMP-2) with bound Pro-macrobodies (Combined focus map)
Components
  • Lysosomal acid glucosylceramidase
  • Lysosome membrane protein 2
  • Nanobody Nb1
  • Nanobody Nb6
KeywordsTRANSPORT PROTEIN / Parkinson / lysosome / Gaucher / GCase / SCARB2 / Pro-macrobody / Glucosylceramide / complex
Function / homology
Function and homology information


regulation of glucosylceramide catabolic process / regulation of carbohydrate catabolic process / regulation of endosome organization / steryl-beta-glucosidase activity / positive regulation of neuronal action potential / beta-glucoside catabolic process / cerebellar Purkinje cell layer formation / aminophospholipid transport / termination of signal transduction / galactosylceramidase ...regulation of glucosylceramide catabolic process / regulation of carbohydrate catabolic process / regulation of endosome organization / steryl-beta-glucosidase activity / positive regulation of neuronal action potential / beta-glucoside catabolic process / cerebellar Purkinje cell layer formation / aminophospholipid transport / termination of signal transduction / galactosylceramidase / galactosylceramidase activity / glucosylceramidase / scavenger receptor binding / lymphocyte migration / glucosylceramide catabolic process / regulation of lysosomal protein catabolic process / response to thyroid hormone / glucosylceramidase activity / sphingosine biosynthetic process / autophagosome organization / lysosomal protein catabolic process / microglial cell proliferation / glucosyltransferase activity / endosome to plasma membrane protein transport / regulation of lysosome organization / regulation of TOR signaling / protein targeting to lysosome / Glycosphingolipid catabolism / microglia differentiation / lipid storage / ceramide biosynthetic process / phosphatidylcholine binding / positive regulation of type 2 mitophagy / : / brain morphogenesis / Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / response to pH / pyramidal neuron differentiation / cargo receptor activity / negative regulation of protein metabolic process / scavenger receptor activity / Transferases; Glycosyltransferases; Hexosyltransferases / lysosome organization / cholesterol binding / neuromuscular process / phosphatidylserine binding / response to dexamethasone / hematopoietic stem cell proliferation / antigen processing and presentation / response to testosterone / Association of TriC/CCT with target proteins during biosynthesis / motor behavior / negative regulation of interleukin-6 production / homeostasis of number of cells / regulation of macroautophagy / establishment of skin barrier / negative regulation of protein-containing complex assembly / cholesterol metabolic process / mitophagy / cell maturation / negative regulation of MAPK cascade / lysosomal lumen / receptor-mediated endocytosis / cellular response to starvation / determination of adult lifespan / respiratory electron transport chain / trans-Golgi network / clathrin-coated endocytic vesicle membrane / sensory perception of sound / positive regulation of neuron projection development / autophagy / negative regulation of inflammatory response / response to estrogen / cellular response to tumor necrosis factor / endocytic vesicle membrane / late endosome membrane / transmembrane signaling receptor activity / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / Cargo recognition for clathrin-mediated endocytosis / T cell differentiation in thymus / Clathrin-mediated endocytosis / protein-folding chaperone binding / virus receptor activity / neuron apoptotic process / proteasome-mediated ubiquitin-dependent protein catabolic process / negative regulation of neuron apoptotic process / lysosome / endosome membrane / signaling receptor binding / Golgi membrane / lysosomal membrane / focal adhesion / endoplasmic reticulum membrane / enzyme binding / endoplasmic reticulum / Golgi apparatus / protein homodimerization activity / extracellular exosome / membrane / plasma membrane
Similarity search - Function
Lysosome membrane protein II / CD36 family / CD36 family / Glycosyl hydrolase family 30, TIM-barrel domain / Glycosyl hydrolase family 30 TIM-barrel domain / Glycosyl hydrolase family 30, beta sandwich domain / Glycosyl hydrolase family 30 beta sandwich domain / Glycoside hydrolase family 30 / Glycoside hydrolase superfamily
Similarity search - Domain/homology
Lysosomal acid glucosylceramidase / Lysosome membrane protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsDobert, J.P. / Schaefer, J.H.S. / Dal Maso, T. / Socher, E. / Versees, W. / Moeller, A. / Zunke, F. / Arnold, P.
Funding support United States, 5items
OrganizationGrant numberCountry
Michael J. Fox FoundationMJFF-17240; MJFF-020706; MJFF-022745 United States
Other governmentInterdisciplinary Center for Clinical Research (IZKF); Jochen-Kalden funding programme N8
Other governmentFonds voor Wetenschappelijk Onderzoek; G031324N
Other governmentVUB Strategic Research Program Financing; SRP95
Other privateFriedrich-Ebert Foundation; no grant number
CitationJournal: Nat Commun / Year: 2025
Title: Cryo-TEM structure of β-glucocerebrosidase in complex with its transporter LIMP-2.
Authors: Jan Philipp Dobert / Jan-Hannes Schäfer / Thomas Dal Maso / Priyadarshini Ravindran / Dustin J E Huard / Eileen Socher / Lisa A Schildmeyer / Raquel L Lieberman / Wim Versées / Arne ...Authors: Jan Philipp Dobert / Jan-Hannes Schäfer / Thomas Dal Maso / Priyadarshini Ravindran / Dustin J E Huard / Eileen Socher / Lisa A Schildmeyer / Raquel L Lieberman / Wim Versées / Arne Moeller / Friederike Zunke / Philipp Arnold /
Abstract: Targeting proteins to their final cellular destination requires transport mechanisms and nearly all lysosomal enzymes reach the lysosome via the mannose-6-phosphate receptor pathway. One of the few ...Targeting proteins to their final cellular destination requires transport mechanisms and nearly all lysosomal enzymes reach the lysosome via the mannose-6-phosphate receptor pathway. One of the few known exceptions is the enzyme β-glucocerebrosidase (GCase) that requires the lysosomal integral membrane protein type-2 (LIMP-2) as a proprietary lysosomal transporter. Genetic variations in the GCase encoding gene GBA1 cause Gaucher's disease (GD) and present the highest genetic risk factor to develop Parkinson's disease (PD). Activators targeting GCase emerge as a promising therapeutic approach to treat GD and PD, with pre-clinical and clinical trials ongoing. In this study, we resolve the complex of GCase and LIMP-2 using cryo-electron microscopy with the aid of an engineered LIMP-2 shuttle and two GCase-targeted pro-macrobodies. We identify helix 5 and helix 7 of LIMP-2 to interact with a binding pocket in GCase, forming a mostly hydrophobic interaction interface supported by one essential salt bridge. Understanding the interplay of GCase and LIMP-2 on a structural level is crucial to identify potential activation sites and conceptualizing novel therapeutic approaches targeting GCase. Here, we unveil the protein structure of a mannose-6-phosphate-independent lysosomal transport complex and provide fundamental knowledge for translational clinical research to overcome GD and PD.
History
DepositionMay 31, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 9, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Lysosome membrane protein 2
B: Lysosomal acid glucosylceramidase
C: Nanobody Nb1
D: Nanobody Nb6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,60221
Polymers128,0774
Non-polymers5,52417
Water48627
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Size exclusion chromatography with western blot analysis of fractions
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Lysosome membrane protein 2 / 85 kDa lysosomal membrane sialoglycoprotein / LGP85 / CD36 antigen-like 2 / Lysosome membrane ...85 kDa lysosomal membrane sialoglycoprotein / LGP85 / CD36 antigen-like 2 / Lysosome membrane protein II / LIMP II / Scavenger receptor class B member 2


Mass: 44862.691 Da / Num. of mol.: 1
Mutation: aa 36-431; N-terminal IgK leader; C-terminal TEV site and 10xHis tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SCARB2, CD36L2, LIMP2, LIMPII / Plasmid: pCMV3_IgK-sLIMP-2-10xHis / Details (production host): pCMV3 backbone / Cell line (production host): HEK 293F / Production host: Homo sapiens (human) / References: UniProt: Q14108
#2: Protein Lysosomal acid glucosylceramidase / Lysosomal acid GCase / Acid beta-glucosidase / Alglucerase / Beta-glucocerebrosidase / Beta-GC / ...Lysosomal acid GCase / Acid beta-glucosidase / Alglucerase / Beta-glucocerebrosidase / Beta-GC / Cholesterol glucosyltransferase / SGTase / Cholesteryl-beta-glucosidase / D-glucosyl-N-acylsphingosine glucohydrolase / Imiglucerase


Mass: 55659.219 Da / Num. of mol.: 1 / Mutation: none
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GBA, GC, GLUC / Plasmid: pCMV3_GBA1 / Details (production host): Sino Biological; #HG12038-UT / Cell line (production host): HEK 293F / Production host: Homo sapiens (human)
References: UniProt: P04062, glucosylceramidase, Transferases; Glycosyltransferases; Hexosyltransferases, EC: 3.2.1.104

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Antibody , 2 types, 2 molecules CD

#3: Antibody Nanobody Nb1


Mass: 14409.816 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Pro-Macrobody; nanobody fused to maltose-binding protein (MBP) via Pro-Pro linker; MBP not resolved and not modeled.
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli MC1061 (bacteria)
#4: Antibody Nanobody Nb6


Mass: 13145.612 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Pro-Macrobody; nanobody fused to maltose-binding protein (MBP) via Pro-Pro linker; MBP not resolved and not modeled.
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli MC1061 (bacteria)

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Sugars , 4 types, 13 molecules

#5: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#6: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#7: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#8: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 31 molecules

#9: Chemical
ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID


Mass: 195.237 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 27 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Lysosomal transporting complex of beta-glucocerebrosidase and lysosomal integral membrane protein 2 (LIMP-2) with two bound Pro-macrobodies.
Type: COMPLEX
Details: Maltose-binding protein (MBP) domains of Pro-macrobodies were not resolved, only nanobody domains are included in the model.
Entity ID: #2, #4, #1, #3 / Source: RECOMBINANT
Molecular weightValue: 0.25 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: hybrid (others)
Buffer solutionpH: 7.4 / Details: 20 mM MES; 150 mM NaCl, pH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMMES; 2-(N-morpholino)ethanesulfonic acidC6H13NO4S1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Monodisperse, complex seperated from monomers via size exclusion chhromatography.
Specimen supportDetails: Protochips; CF-1.2/1.3-3Cu-50 / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10550
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategoryFitting-ID
1cryoSPARC4.2particle selection
2EPUimage acquisition
3UCSF ChimeraX1.7.1masking
4cryoSPARC4.2masking
5cryoSPARC4.2CTF correction
6CootWinCoot 0.9.8.7model fitting1
7UCSF ChimeraX1.7.1model fitting1
8PHENIXmodel refinement
9MODELLERother
10cryoSPARCclassification
11cryoSPARC3D reconstruction
15PHENIX1.20.1_4487model refinement1
16CootWinCoot 0.9.8.7model fitting2
17UCSF ChimeraX1.7.1model fitting2
18PHENIX1.20.1_4487model refinement2
19CootWinCoot 0.9.8.7model fitting3
20UCSF ChimeraX1.7.1model fitting3
21PHENIX1.20.1_4487model refinement3
25PHENIX1.20.1_44873D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 397385 / Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1FLEXIBLE FITREALInitial fitting with ChimeraX, manual flexible fitting with Coot, refinement with Phenix
2FLEXIBLE FITREALInitial fitting with ChimeraX, manual flexible fitting with Coot, refinement with Phenix
3RIGID BODY FITREALInitial fitting with ChimeraX, manual flexible fitting with Coot, refinement with Phenix
Atomic model building

Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-ID 3D fitting-IDAccession codeChain-IDChain residue rangeDetailsInitial refinement model-IDPdb chain residue range
16TN1

6tn1

AAA16TN1AAA1-497GCase11-497
24Q4F

4q4f

A24Q4FA37-430LIMP-2237-430
39ENA

9ena

39ENANanobody Nb1; Unpublished3

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